Historically, the detection and identification of bacteria, mycobacteria, yeasts, and molds have relied primarily on their morphologic and phenotypic properties. This approach is imprecise and slow for many clinically significant microbes. For this reason, we have explored alternative methods, such as Sanger sequencing, pyrosequencing, and mass spectroscopy, for the detection and identification of selected organisms. Sanger sequencing is used routinely for the identification of bacteria including Nocardia, mycobacteria, and molds; however, the procedure is expensive and time-consuming. Pyrosequencing provides an attractive alternative because amplification of target gene sequences and analysis of the amplified product is rapid and less expensive. To test this methodology, we used pyrosequencing to identify 133 isolates of clinically relevant non-dematiaceous yeasts. These included 97 ATCC strains (42 type strains), 7 strains from other national culture collections, and 29 clinical isolates. Isolates belonged to the following genera: Candida (18 species), Trichosporon (10), Cryptococcus (7), Malassezia (3), Rhodotorula (2), Geotrichum (1), Blastoschizomyces (1) and Kodamaea (1). Amplicons were obtained of a hypervariable ITS region and analyzed using pyrosequencing technology. The data were evaluated by a BLAST search against the GenBank database and correlated with data obtained by conventional cycle sequencing of the ITS1-5.8S-ITS2 region. Sanger sequencing identified (78.9%) of the isolates to the species level; pyrosequencing technology identified 69.1%. In 90.1% of all the strains tested, identification results of both sequencing methods were identical. Most Candida isolates, the most common clinically, were identified to the species level by pyrosequencing. Trichosporon species and some Cryptococcus cannot be differentiated at the species level. We concluded from these studies that pyrosequencing could be used for the reliable identification of most commonly isolated non-dematiaceous yeasts, with a reduction of cost per identification compared to conventional sequencing. [unreadable] [unreadable] We also have evaluated a proteomic approach, specifically matrix-assisted laser desorption-time of flight mass spectrometery (MALDI-TOF-MS), for the identification of clinically important microbes. MALDI-TOF-MS provides a reproducible spectral pattern based on the mass/charge (m/z) ratio of ionized proteins. While robust spectral libraries exist for many bacteria, there remains a need to enhance the representation of clinically important yeast species. Therefore, 100 ATCC strains (47 species) were evaluated for database construction and clinical isolates were used as challenge organisms. Colony material was extracted with 1:1 Formic Acid (70%):Acetonitrile and 1l of the extracted sample was analyzed using -cyano-4-hydroxycinnamic acid as the matrix. Spectra were produced on an UltraFlex MALDI-TOF spectrometer (Bruker Daltonics, Germany). MALDI-TOF spectrum were obtained for 100 yeast isolates and used to create a database within the BioTyper 2.0 software package (Bruker). Unique spectral profiles were observed for all species except Cryptococcus luteolus (N=4) and Trichosporin asahii (N=2). Patterns were reproducible for all isolates representing multiple species including: Candida dubliniensis (n=5), C. pintolopesii (n=5), C. catenulate (n=3), C. zeylanoidea (n=3), C. kefyr (n=4), C. lipolytica (n=5), C. rugosa (n=4), Geotrichium capitatum (n=2), Trichosporon inkin (n=4), T. ovoides (n=4), Cryptococcus laurentii (n=5), C. uniguttulatus (n=2), C. neoformans (n=10), C. terreus (n=3). The following challenge clinical isolates were identified correctly: C. albicans (n=5), C. glabrata (n=4), C. tropicalis (n=5), C. parapsilosis (n=5), C. krusei (n=2) and Rhodotorula sp. (n=1). Validation of the database with additional clinical isolates is ongoing. The turnaround time for identification was <30 minutes. These preliminary studies support the use of MALDI TOF MS as a quick and accurate method for the identification of most clinical isolates of yeast. Additional studies are also underway to assess the performance of MALDI TOF MS for identification of Nocardia, mycobacteria, and nonfermentative bacteria, as well as direct identification of microbes in specimens such as blood cultures.